Ag85B And 19kDa Signal Peptide Mediated Bovine Viral Diarrhea Virus E₀- E₂ Fusion Gene Expression And Immunogenicity Validation In Recombinant BCG

Bovine viral diarrhea virus(BVDV), a Flaviviridae pestivirus, reduces productivity, abortion, infertility, and the fatal mucosal disease. E0 possessing RNase activity and E2 membrane-anchored in viral replication and pathogenesis are the major protective protein, which stimulate the organism to produce the neutralizing antibody.These different forms of display and the expression of foreign antigen by BCG cells can be influenced by signal peptide. The signal peptide of Ag85 B and 19 kDa was researched in recombinant BCG(rBCG).The signal peptide including Ag85 B and 19 kDa was fused with the E0 gene of C24 V standard strain, E2 truncated gene of changchun184 and the E0-E2 fusion gene by overlapping extension. The gene was subcloned to vector pMV261, while the rBCG was constructed. By heat-induced at 45℃, the expression of rBCG was analyzed by SDS-PAGE and Western blot. The mice were injected by rBCG, while BVDV antibody, levels of CD4+, CD8+ and IL-4, IL-10, IL-12 and IFN-γ were detected.In this research, rBCG-Ag85B-E0, rBCG-19kDa-E0, rBCG-Ag85B-E2, rBCG-19kDa-E2, rBCG-Ag85B-E0-E2 and rBCG-19kDa-E0-E2 was constructed. The results of SDS-PAGE has shown that the protein induced by rBCG was distributed in deposition. The results have shown that the anti-BVDV antibodies were induced by rBCG. The level of CD4+ was dominant, while the level of CD8+ of rBCG induced by 19 k Da was higher. Moreover, due to the level of IFN-γ was markly higher than other level of cytokine, BCG was mediated to result in the Th1 immune response, while the level of Cytokines in BCG was higher than rBCG. It demonstrated that the positive inductive effect on the humoral and cellular immunity wad induced by rBCG, and the rBCG mediated by 19 kDa can induce the production of immunogenicity.This study has demonstrated that the immunogenicity can be improved by the rBCG mediated by signal peptide and the rBCG mediated by 19 kDa was better than others, which laid the goundation for the study of engineering vaccine against BVDV.